Specneuzhenide improves bleomycin-induced pulmonary fibrosis in mice via AMPK-dependent reduction of PD-L1.

BACKGROUND: Pulmonary fibrosis (PF) is an escalating global health issue, characterized by rising rates of morbidity and mortality annually. Consequently, further investigation of potential damage mechanisms and potential preventive strategies for PF are warranted. Specnuezhenide (SPN), a prominent secoiridoid compound derived from Ligustrum lucidum Ait, exhibits anti-inflammatory and anti-oxidative capacities, indicating the potential therapeutic actions on PF. However, the underlying mechanisms of SPN on PF remain unclear. PURPOSE: This work was aimed at investigating the protective actions of SPN on PF and the potential mechanism. METHODS: In vivo, mice were administrated with bleomycin (BLM) to establish PF model. PF mice were treated with SPN (45/90 mg/kg) by gavage. In vitro, we employed TGF-ß1 (10 ng/mL)-induced MLE-12 and PLFs cells, which then were treated with SPN (5, 10, 20 µM). DARTS assay, biofilm interference experiment and molecular docking were performed to investigate the molecular target of SPN. RESULTS: In vivo, we found SPN treatment improved survival rate, alleviated pathological changes through reducing BLM-induced extracellular matrix (ECM) deposition, as well as BLM-induced epithelial-mesenchymal transition (EMT). In vitro, SPN inhibited EMT and lung fibroblast transdifferentiation. Mechanistically, SPN activated the AMPK protein to decrease the abnormally high level of PD-L1. Furthermore, the compound C, known as an AMPK inhibitor, exhibited a significant hindrance to the inhibition of SPN on TGF-ß1-caused fibroblast transdifferentiation and proliferation. This outcome could be attributed to the fact that compound C could eliminate the inhibitory effects of SPN on PD-L1 expression. Interestingly, DARTS assay, biofilm interference experiment and molecular docking results all indicated that SPN could bind to AMPK, which suggested that SPN might be a potential agonist targeting AMPK protein. CONCLUSION: Altogether, the results in our work illustrated that SPN promoted AMPK-dependent reduction of PD-L1 protein, contributing to the inhibition of fibrosis progression. Thus, SPN may represent a potential AMPK agonist for PF treatment.

Erzhiwan Ameliorates Restraint Stress- and Monobenzone-Induced Depigmentation in Mice by Inhibiting Macrophage Migration Inhibitory Factor and 8-Hydroxy-2-Deoxyguanosine.

Purpose: Vitiligo is an autoimmune disease that results in the loss of epidermal melanocytes. The treatments for patients with vitiligo remain lacking. Erzhiwan (EZW), a traditional Chinese Medicine composed of Ligustri Lucidi Fructus and Ecliptae Herba, was used to ameliorate depigmentation since ancient China. This study aims to investigate the effect of EZW on vitiligo-related depigmentation. Methods: A vitiligo-related depigmentation mouse model was induced by monobenzone and restraint stress. The experimental depigmentation mice were treated with EZW. Histological observation of skin was conducted. Cutaneous oxidative damage and inflammation were determined. A network pharmacology analysis was carried out. Results: EZW reduced depigmentation score (p<0.01), cutaneous inflammatory infiltration (p<0.01), and CD8α-positive expression (p<0.01), and increased cutaneous melanin content in experimental depigmentation mice. EZW reduced stress reaction in experimental depigmentation mice (p<0.01). EZW inhibited 8-hydroxy-2-deoxyguanosine (8-OHdG)-related DNA oxidative damage in the skin (p<0.05, p<0.01). In addition, EZW reduced cutaneous macrophage migration inhibitory factor (MIF)-CD74-NF-κB signaling (p<0.01). The network pharmacology analysis demonstrated that EZW regulated necroptosis, apoptosis, and FoxO signaling pathways in vitiligo. An in vitro experiment showed that the main ingredient of EZW, specnuezhenide, protected against monobenzone and MIF-induced cell death in HaCaT cells (p<0.01). Conclusion: EZW ameliorates restraint stress- and monobenzone-induced depigmentation via the inhibition of MIF and 8-OHdG signaling. The findings provide a data basis of an utilization of EZW in vitiligo.

Gut microbiota-based pharmacokinetic-pharmacodynamic study and molecular mechanism of specnuezhenide in the treatment of colorectal cancer targeting carboxylesterase.

Specnuezhenide (SNZ) is among the main components of Fructus Ligustri Lucidi, which has anti-inflammation, anti-oxidation, and anti-tumor effect. The low bioavailability makes it difficult to explain the mechanism of pharmacological effect of SNZ. In this study, the role of the gut microbiota in the metabolism and pharmacokinetics characteristics of SNZ as well as the pharmacological meaning were explored. SNZ can be rapidly metabolized by the gut microbiome, and two intestinal bacterial metabolites of SNZ, salidroside and tyrosol, were discovered. In addition, carboxylesterase may be the main intestinal bacterial enzyme that mediates its metabolism. At the same time, no metabolism was found in the incubation system of SNZ with liver microsomes or liver homogenate, indicating that the gut microbiota is the main part involved in the metabolism of SNZ. In addition, pharmacokinetic studies showed that salidroside and tyrosol can be detected in plasma in the presence of gut microbiota. Interestingly, tumor development was inhibited in a colorectal tumor mice model administered orally with SNZ, which indicated that SNZ exhibited potential to inhibit tumor growth, and tissue distribution studies showed that salidroside and tyrosol could be distributed in tumor tissues. At the same time, SNZ modulated the structure of gut microbiota and fungal group, which may be the mechanism governing the antitumoral activity of SNZ. Furthermore, SNZ stimulates the secretion of short-chain fatty acids by intestinal flora in vitro and in vivo. In the future, targeting gut microbes and the interaction between natural products and gut microbes could lead to the discovery and development of new drugs.

Specnuezhenide Ameliorates Age-Related Hepatic Lipid Accumulation via Modulating Bile Acid Homeostasis and Gut Microbiota in D-Galactose-Induced Mice.

Age-related hepatic lipid accumulation has become a major health problem in the elderly population. Specnuezhenide (SPN) is a major active iridoid glycoside from an edible herb Fructus Ligustri Lucidi, which is commonly used for preventing age-related diseases. However, the beneficial effects of SPN on age-related liver injury remain unknown. This study aimed to reveal the effect of SPN on age-related hepatic lipid accumulation and the underlying mechanism. D-galactose (D-gal)-induced aging mice were treated with vehicle or SPN for 12 weeks. Treatment of SPN decreased lipid accumulation and inflammation in the liver of D-gal-induced mice. Untargeted and targeted metabolomics showed that the SPN could regulate the bile acid (BA) synthesis pathway and restore the BA compositions in serum, livers, and feces of the D-gal-induced mice. Furthermore, SPN enhanced the protein and mRNA levels of hepatic BAs synthesis enzymes cytochrome P45027A1, cytochrome P4507A1, cytochrome P4507B1, and cytochrome P4508B1. Meanwhile, SPN alleviated D-gal-induced gut dysbiosis and reversed the proportions of microbes associated with bile salt hydrolase activity, including Lactobacillus, Ruminiclostridium, and Butyrivibrio. Our study revealed that SPN attenuated age-related hepatic lipid accumulation by improving BA profiles via modulating hepatic BA synthesis enzymes and gut microbiota.

Effect and Mechanism of Specnuezhenide on Chemotherapy-induced Myelosuppression.

OBJECTIVE: This study aimed to investigate the therapeutic effect of Specnuezhenide on myelosuppression induced by chemotherapy and clarify its mechanism. METHODS: In this study, we measured peripheral blood cells, thymus index, spleen index, bone marrow nucleated cells (BMNCs), and the number of cell colonies counted in vitro by hematopoietic progenitor cells (HPCs) to determine the effect of SPN on cyclophosphamide (CTX)-induced myelosuppression. The alterations in the expression of relevant proteins, the cell cycle, and cytokines associated with hematopoietic cells were examined to better understand how it works. RESULTS: In the cyclophosphamide-induced mouse model, our study discovered that SPN can increase the number of peripheral blood cells and BMNCs after treatment, increase the thymus index and decrease the spleen index, and promote the proliferation and differentiation of HPCs. SPN can improve the production of cultured colonies in vitro, reduce the level of hematopoietic factors in vivo, regulate the proportion of G0/G1 phase cells, and promote the normal growth and development of cells. SPN can increase the expression levels of key proteins MEK and p-ERK in the MAPK signaling pathway, which may be one of the important mechanisms for improving myelosuppression. CONCLUSION: SPN can enhance the hematological and immunological functions of myelosuppressionmice, and it is hypothesized that SPN is extremely helpful to the hematopoietic and immune functions of tumor patients following chemotherapy. SPN might be used to treat myelosuppression. Additionally, high doses of SPN have a stronger therapeutic effect than low levels of SPN.

Inhibitory effect of specnuezhenide on high glucose-induced human retinal microvascular endothelial cell injury and its mechanism / 中华实验眼科杂志

Objective:To investigate the effect of specnuezhenide on high glucose-induced human retinal microvascular endothelial cells (hRMECs) injury and its mechanism.Methods:The hRMECs were divided into a normal control group cultured in a culture medium containing 5.5 mmol/L glucose, a hypertonic group cultured in a culture medium containing 5.5 mmol/L glucose + 24.5 mmol/L mannitol, a high glucose group cultured in a culture medium containing 30 mmol/L glucose, as well as high glucose+ low-, medium-, and high-dose specnuezhenide groups cultured in culture media containing 30 mmol/L glucose + 25, 50, 100 μmol/L specnuezhenide for 24 hours, respectively.In addition, hRMECs were divided into a high glucose+ small interfering RNA-negative control (si-NC) group cultured in a culture medium containing 30 mmol/L glucose, a high glucose+ si-forkhead box O4 (FOXO4) group cultured in a culture medium containing 30 mmol/L glucose, a high glucose+ specnuezhenide+ pcDNA group cultured in a culture medium containing 100 μmol/L specnuezhenide + 30 mmol/L glucose, and a high glucose+ specnuezhenide+ pcDNA-FOXO4 group cultured in a culture medium containing 100 μmol/L specnuezhenide+ 30 mmol/L glucose for 24 hours after transfection by corresponding reagents.Cell apoptosis was detected by flow cytometry.The malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity in cells were detected by the thiobarbituric acid method and xanthine oxidase method, respectively.The concentrations of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the cell culture supernatant were detected by enzyme linked immunosorbent assay.The relative expression level of FOXO4 protein in cells was determined by Western blot.Results:The apoptosis rates of normal control group, hypertonic group, high glucose group, high glucose+ low-, medium- and high-dose specnuezhenide groups were (7.32±0.72)%, (7.44±0.70)%, (23.96±1.32)%, (19.84±1.09)%, (14.13±0.85)% and (9.84±0.70)%, respectively.There were significant differences in cell apoptosis rate, MDA concentration, SOD activity, the concentration of IL-1β, the concentration of TNF-α, and the relative expression level of FOXO4 protein among the six groups ( F=498.545, 1 186.693, 516.629, 654.247, 638.238, 472.655; all at P<0.001). Compared with high glucose group, the apoptosis rate, MDA concentration, IL-1β and TNF-α concentration, FOXO4 protein expression level were significantly decreased in high glucose+ low-, medium- and high-dose specnuezhenide groups, and SOD activity was significantly increased in a dose-dependent manner.Compared with high glucose+ si-NC group, the expression level of FOXO4 protein, cell apoptosis rate, MDA concentration, IL-1β and TNF-α mass concentrations were decreased in high glucose + si-FOXO4 group, while the SOD activity was increased.Compared with high glucose+ specnuezhenide+ pcDNA group, the apoptosis rate, MDA concentration, IL-1β and TNF-α concentrations, FOXO4 protein expression level of hRMECs in high glucose+ specnuezhenide+ pcDNA-FOXO4 group were significantly increased, and SOD activity was significantly decreased (all at P<0.05). Conclusions:Specnuezhenide can protect hRMECs from high glucose-induced apoptosis, oxidative stress and inflammatory response by down-regulating FOXO4.

Specnuezhenide suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis.

Diabetes osteoporosis is a chronic complication of diabetes mellitus (DM) and is associated with osteoclast formation and enhanced bone resorption. Specnuezhenide (SPN) is an active compound with anti-inflammatory and immunomodulatory properties. However, the roles of SPN in diabetic osteoporosis remain unknown. In this study, primary bone marrow macrophages (BMMs) were pretreated with SPN and were stimulated with receptor activator of nuclear factor kappa B ligand (RANKL; 50 ng/mL) to induce osteoclastogenesis. The number of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. The protein levels of cellular oncogene fos/nuclear factor of activated T cells c1 (c-Fos/NFATc1), nuclear factor kappa-B (NF-κB), and mitogen-activated protein kinases (MAPKs) were evaluated by western blot analysis. NF-κB luciferase assays were used to examine the role of SPN in NF-κB activation. The DM model group received a high-glucose, high-fat diet and was then intraperitoneally injected with streptozotocin (STZ). Micro-CT scanning, serum biochemical analysis, histological analysis were used to assess bone loss. We found that SPN suppressed RANKL-induced osteoclast formation and that SPN inhibited the expression of osteoclast-related genes and c-Fos/ NFATc1. SPN inhibited RANKL-induced activation of NF-κB and MAPKs. In vivo experiments revealed that SPN suppressed diabetes-induced bone loss and the number of osteoclasts. Furthermore, SPN decreased the levels of bone turnover markers and increased the levels of runt-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), calcium (Ca) and phosphorus (P). SPN also regulated diabetes-related markers. This study suggests that SPN suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis, and provides an experimental basis for the treatment of diabetic osteoporosis.

Specnuezhenide reduces carbon tetrachloride-induced liver injury in mice through inhibition of oxidative stress and hepatocyte apoptosis.

OBJECTIVES: This study aimed to investigate the hepatoprotective effects of specnuezhenide against carbon tetrachloride (CCl4)-induced liver injury in mice. METHODS: Male C57BL/6 mice were intraperitoneally injected with 10 ml/kg body weight of CCl4 (0.5% diluted in arachis oil) for acute liver injury after oral administration of specnuezhenide for 7 days. Twenty-four hours after the final CCl4 injection, mice were euthanized and plasma and liver samples were collected. KEY FINDINGS: The results showed that specnuezhenide markedly and dose-dependently reduced serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) activity and relative liver weight, as well as ameliorated histopathological damage caused by CCl4 and decreased malondialdehyde (MDA) levels, and increased the activity of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Moreover, specnuezhenide promoted the expression and nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and increased the mRNA and protein expression of Nrf2 signalling-related genes heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC) and NAD(P)H:quinone oxidoreductase 1 (NQO1). Finally, TUNEL staining and immunohistochemistry indicated that specnuezhenide prevented CCl4-induced hepatocytic apoptosis by up-regulating B-cell lymphoma 2 (Bcl-2) expression and downregulating Bcl-2-associated X (Bax) expression. CONCLUSIONS: Specnuezhenide reduced CCl4-induced liver injury in mice by inhibiting oxidative stress via activation of Nrf2 signalling and decreasing hepatocyte apoptosis.

Comparison of Zhuyao and Douchi Ligustri Lucidi Fructus Based on Quality Investigation of Commercial Samples / 中国实验方剂学杂志

ObjectiveTo investigate the quality of Ligustri Lucidi Fructus in the market， and the moisture， extract， determination of Zhuyao and Douchi Ligustri Lucidi Fructus were compared to increase the utilization rate of Ligustri Lucidi Fructus. MethodThe properties， moisture， total ash， alcohol-soluble extract content and thin layer chromatography （TLC） identification were determined by the methods of Ligustri Lucidi Fructus included in the 2020 edition of Chinese Pharmacopoeia， and high performance liquid chromatography （HPLC） fingerprint and determination of specnuezhenide and salidroside were established with the mobile phase of 0.2% phosphoric acid aqueous solution （A）-acetonitrile （B） （0-70 min， 92%-65%A） for gradient elution， and the detection wavelength of 220 nm at 0-14 min and 225 nm at 14-70 min. The two different characters of Ligustri Lucidi Fructus were comprehensively compared by the above indicators. ResultExcept for one batch which did not meet the requirements due to the quality of harvesting， the other 12 batches of samples all met the requirements of the 2020 edition of Chinese Pharmacopoeia， but there were two different characters. Comparing the two different characters of Ligustri Lucidi Fructus， it is found that the moisture， total ash， extract， salidroside and specnuezhenide contents of Zhuyao samples were 2.22%-5.19%， 3.91%-4.49%， 32.56%-40.95%， 0.073%-0.170% and 1.45%-4.14%， and these values of Douchi samples were 3.57%-5.61%， 3.65%-4.44%， 41.31%-46.70%， 0.041%-0.067% and 3.01%-4.20%， respectively. ConclusionThe contents of extract and specnuezhenide of Douchi Ligustri Lucidi Fructus are mostly higher than those of Zhuyao Ligustri Lucidi Fructus， while the content of salidroside is lower than that of Zhuyao samples， and there are no significant differences in moisture， TLC identification and total ash content. Based on the above research， if the main purpose is to extract salidroside， it is recommended to choose Zhuyao Ligustri Lucidi Fructus. If the main purpose is to use Ligustri Lucidi Fructus as medicine， it is recommended to choose Douchi Ligustri Lucidi Fructus.

Protective effect of Specnuezhenide on islet ß cell of rats with gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) refers to diabetes mellitus and impaired glucose tolerance first diagnosed during pregnancy without previous history of the disease. Usually, GDM has a high risk of inducing type 2 diabetes mellitus. The incidence of GDM is increasing year by year worldwide, and it seriously affects the quality of life of patients, and increases the risk to pregnancy. Specnuezhenide (SPZ) is a characteristic active component of Ligustrum lucidum, a plant which exerts a variety of pharmacological effects. In this study, the protective effect of SPZ on ß cells in gestational diabetes mellitus rats was investigated in a rat model of gestational diabetes. Based on oral glucose tolerance test, ELISA, qRT-PCR and western blotting assays, It was found that SPZ effectively improved blood sugar control and glucose tolerance in gestational diabetic rats, inhibited inflammation in islet tissue, and reduced inflammation-mediated insulin resistance.

Protective Effects of Specneuzhenide on Renal Injury in Rats with Diabetic Nephropathy.

BACKGROUND: We aim to investigate the protective effects and potential mechanisms in specneuzhenide (SPE) on renal injury in rats with diabetic nephropathy (DN). RESULTS: SPE could inhibit the decrease of body weight compared with the model group (P<0.05), and trigger improvement in the renal index (P<0.05). High dose and low dose SPE could trigger a significant decrease in serum IL1ß, IL-6 and TNF-α compared with the model group (P<0.05). SPE could attenuate the glomerular lesions in DN rats. SPE induced up-regulation of podocin and CD2AP (P<0.05). CONCLUSION: SPE showed protective effects on renal injury through attenuating the pathological injury and urine protein. This process may be closely related to the modulation of CD2AP and podocin expression.

A validated LC-MS/MS method for the determination of specnuezhenide and salidroside in rat plasma and its application to a pharmacokinetic study.

The aim of this study was to establish and validate a rapid and sensitive LC-MS/MS method for the simultaneous determination of specnuezhenide and its bioactive metabolite salidroside in rat plasma. Protein precipitation was carried out and the analytes were separated on a Waters Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm). A mobile phase consisting of acetonitrile and 0.1% formic acid aqueous solution was used for elution under gradient conditions at a flow rate of 0.4 mL/min. Quantification was performed in the negative multiple reaction monitoring mode with precursor-to-product transitions at m/z 685.2 → 453.1 for specnuezhenide, m/z 229.3 → 119.0 for salidroside and 493.2 → 147.1 for the internal standard. The method showed good linearity, accuracy, precision and stability in the range 0.5-500.0 ng/mL for specnuezhenide and salidroside. The values of the matrix effect were within the range of 100.02-111.87% for both analytes, while the mean extraction recovery was within the range 64.19-78.26%. The intra- and inter-day precisions (RSD) were <13.49% and the accuracy (RR) ranged from 93.59 to 102.24%. This study was successfully utilized for the pharmacokinetic study of specnuezhenide in rats after oral and intravenous administration. The oral bioavailability of specnuezhenide was 1.93%.

Specnuezhenide Decreases Interleukin-1ß-Induced Inflammation in Rat Chondrocytes and Reduces Joint Destruction in Osteoarthritic Rats.

As a chronic disease, osteoarthritis (OA) leads to the degradation of both cartilage and subchondral bone, its development being mediated by proinflammatory cytokines like interleukin-1ß. In the present study, the anti-inflammatory effect of specnuezhenide (SPN) in OA and its underlying mechanism were studied in vitro and in vivo. The results showed that SPN decreases the expression of cartilage matrix-degrading enzymes and the activation of NF-κB and wnt/ß-catenin signaling, and increases chondrocyte-specific gene expression in IL-1ß-induced inflammation in chondrocytes. Furthermore, SPN treatment prevents the degeneration of both cartilage and subchondral bone in a rat model of OA. To the best of our knowledge, this study is the first to report that SPN decreases interleukin-1ß-induced inflammation in rat chondrocytes by inhibiting the activation of the NF-κB and wnt/ß-catenin pathways, and, thus, has therapeutic potential in the treatment of OA.

[Content change of main components in processed Ligustrum lucidum fruits of different processing time and their characteristic quality standard based on UPLC technology].

To investigate the dynamic change law of the main components in Ligustri Lucidi Fructus during the wine-steaming process and attempt to establish the characteristic quality standard of wine-steamed Ligustri Lucidi Fructus by determining the content of salidroside and specnuezhenide using Ultra Performance Liquid Chromatography (UPLC) technology at different processing time points (12, 15, 18, 21, 24 h). The chromatographic separation was performed on Waters Acquity UPLC®BEH C18 column (2.1 mm×50 mm, 1.7 µm) with acetonitrile and 0.2% formic acid aqueous solution as the mobile phase for gradient elution; and the detection wavelength was set at 280 nm; the flow rate was 0.5 mL·min⁻¹, and the column temperature was set at 40 °C. The results showed that the two components were well separated in the above conditions. The salidroside and specnuezhenide showed a good linear relationship within the range of 10.19-326 ng and 49.53-1 585 ng, respectively. Their average recovery was 103.4% and 101.7% with RSD of 0.81% and 0.79%, respectively. With the extension of processing time, the content of specnuezhenide was decreased, while salidroside was gradually increased. For the 27 batches of Ligustri Lucidi Fructus, the content of salidroside was between 0.042 5% and 0.192 4%, and that of specnuezhenide was between 0.829 7% and 5.218 0%. While for the 25 batches of wine-steamed Ligustri Lucidi Fructus, the content of the first one was between 0.229 2% and 1.045 8%, and the latter one was between 0.743 8% and 3.645 4%. As compared with Ligustri Lucidi Fructus, the ratio of specnuezhenide to salidroside was significantly decreased in the wine-steamed Ligustri Lucidi Fructus. According to the experimental results, the quality standard of Ligustri Lucidi Fructus is tentatively fixed as follows: the content of specnuezhenide shall not be less than 0.80%, and the ratio of specnuezhenide content/salidroside content (Sp/Sa) should not be smaller than 15. As for the wine-steamed ones, the content of salidroside should not be less than 0.20%, and specnuezhenide content should not be less than 0.70%; Sp/Sa should not be greater than 8. The method established in this study is simple and reliable, which could be used for the content detection of salidroside and specnuezhenide in Ligustri Lucidi Fructus samples. The characteristic quality standard established in this study could be used to distinguish the Ligustri Lucidi Fructus and wine-steamed Ligustri Lucidi Fructus.

Content change of main components in processed Ligustrum lucidum fruits of different processing time and their characteristic quality standard based on UPLC technology / 中国中药杂志

To investigate the dynamic change law of the main components in Ligustri Lucidi Fructus during the wine-steaming process and attempt to establish the characteristic quality standard of wine-steamed Ligustri Lucidi Fructus by determining the content of salidroside and specnuezhenide using Ultra Performance Liquid Chromatography (UPLC) technology at different processing time points (12, 15, 18, 21, 24 h). The chromatographic separation was performed on Waters Acquity UPLC®BEH C₁₈ column (2.1 mm×50 mm, 1.7 μm) with acetonitrile and 0.2% formic acid aqueous solution as the mobile phase for gradient elution; and the detection wavelength was set at 280 nm; the flow rate was 0.5 mL·min⁻¹, and the column temperature was set at 40 °C. The results showed that the two components were well separated in the above conditions. The salidroside and specnuezhenide showed a good linear relationship within the range of 10.19-326 ng and 49.53-1 585 ng, respectively. Their average recovery was 103.4% and 101.7% with RSD of 0.81% and 0.79%, respectively. With the extension of processing time, the content of specnuezhenide was decreased, while salidroside was gradually increased. For the 27 batches of Ligustri Lucidi Fructus, the content of salidroside was between 0.042 5% and 0.192 4%, and that of specnuezhenide was between 0.829 7% and 5.218 0%. While for the 25 batches of wine-steamed Ligustri Lucidi Fructus, the content of the first one was between 0.229 2% and 1.045 8%, and the latter one was between 0.743 8% and 3.645 4%. As compared with Ligustri Lucidi Fructus, the ratio of specnuezhenide to salidroside was significantly decreased in the wine-steamed Ligustri Lucidi Fructus. According to the experimental results, the quality standard of Ligustri Lucidi Fructus is tentatively fixed as follows: the content of specnuezhenide shall not be less than 0.80%, and the ratio of specnuezhenide content/salidroside content (Sp/Sa) should not be smaller than 15. As for the wine-steamed ones, the content of salidroside should not be less than 0.20%, and specnuezhenide content should not be less than 0.70%; Sp/Sa should not be greater than 8. The method established in this study is simple and reliable, which could be used for the content detection of salidroside and specnuezhenide in Ligustri Lucidi Fructus samples. The characteristic quality standard established in this study could be used to distinguish the Ligustri Lucidi Fructus and wine-steamed Ligustri Lucidi Fructus.

Iridoid glycoside extraction technology and anti-thrombotic activity of Osmanthus fragrans seeds / 中草药

Objective: To optimize the extraction technology of two major iridoid glucosides (specnuezhenide and nuezhenoside G13) from four kinds of Osmanthus fragrans (OF) seeds, and to evaluate the anti-thrombotic activity of OF seeds. Methods: The orthogonal-test experiment was employed to optimize the parameters including ethanol concentration, liquid-material ratio, and extraction time for three extraction methods (ultrasonic extraction, reflux extraction, and microwave extraction). The extraction yield, content, and total peak area of iridoid glucosides were selected for weighted analysis to determine the best extraction method and technology. Additionally, an anti-thrombotic zebra fish model was established for biological evaluation of OF seeds. Results: Microwave extraction was the best method for iridoid glucosides extraction with the optimal conditions of ethanol concentration 55%, material-liquid ratio 1∶10, and microwave time 15 min. HPLC analysis showed that there was no significant difference in chemical composition among the four kinds of OF seeds. In zebra fish biological screening model, OF seeds displayed a weak inhibitory effect on the growth of thrombus and exhibited a pericardial edema effect in high dose-treated group. Conclusion: In this paper, extraction technology of two iridoid glucosides from four different kinds of OF seeds and preliminary anti-thrombotic activity evaluation of OF seeds were investigated. These results can provide the reference for further development and utilization of the agricultural waste of OF seeds.

Simultaneous determination of six constituents in Yitai Capsules by HPLC / 中成药

AIM To establish an HPLC method for the simultaneous content determination of ginsenoside Re,ginsenoside Rg1,ginsenoide Rb1,specnuezhenide,calycosin-7-O-β-D-glucoside and oleanolic acid in Yitai Cap sules (Ginseng Radix et Rhizoma,Astragali Radix,Ligustri lucidi Fructus,etc.).METHODS The analysis of 70％ ethanol extract of this drug was performed on a 25 ℃ thermostatic Luna C18 column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of methanol-O.2％ phosphoric acid flowing at 1.0 mL/minin a gradient elution manner,and the detection wavelength was set at 203 nm.RESULTS Six constituents showed good linear relationships within their own ranges (r ＞ 0.999 0),whose average recoveries were 95.58％-102.12％ with the RSDs of 0.82％-1.73％.CONCLUSION This simple and stable method can be used for the rapid quality control of Yitai Capsules.

Quality stability of Ligustri Lucidi Fructus / 中国药学杂志

OBJECTIVE: To study the change rule of specnuezhenide content and antoxidant activity of Ligustri Lucidi Fructus with temperature and time, and determine its validity period. METHODS: Dynamic parameters were established under constant temperature, and specnuezhenide content of Ligustri Lucidi Fructus was determined by HPLC after accelerated test by classical constant temperature method. The shelf life of Ligustri Lucidi Fructus at 25℃ were determined by Arrhenius theory. The antioxidant activities of different samples of Ligustri Lucidi Fructus were evaluated by ABTS assay. RESULTS: The shelf life of Ligustri Lucidi Fructus was forecasted to be 1.83 years at 25℃. The antioxidant activitiy of Ligustri Lucidi Fructus showed a trend of decline after rising first. CONCLUSION: High temperature and storage time would affect the chemical composition of Ligustri Lucidi Fructus, so Ligustri Lucidi Fructus should be kept in the shade for no more than 1.83 years for a good therapeutic effect.

Studies on quality specification of Zishen Yangyin Granules / 中草药

Objective: To establish methods of qualitative identification and quantitative determination for Zishen Yangyin Granules (ZYG). Methods: The TLC method was used to identify the herb by mixture of chloroform-methanol (31) as a developing solvent on high performance silica gel precoated plate (HSGF254) and using 5% vanillic aldehyde sulfuric acid as a chromogenic reagent for qualitative identification of Corni Fructus; TLC identification of Eclipta prostrata, Alismatis Rhizoma, and Moutan Cortex was performed on high performance silica gel precoated plate (HSGF254) with petroleum ether (60-90 ℃)-chloroform-ethyl acetate (312) as developing solvent. The same developing method was used to identify E. prostrata, A. Rhizoma, and paneol in M. Cortex of ZYG by different detected method at the same time. The contents of morroniside, loganin, hyperoside, specnuezhenide, and paeonol were analyzed by high performance liquid chromatography on C18 column (250 mm × 4.6 mm, 5 μm) with the mobile phase of acetonitrile-0.1% trifluoroacetic acid by gradient elution. The detection wavelength was set at 254 nm. Results: Morroniside and loganin were used to identify C. Fructus in ZYG, paeonol in M. Cortex can be identified at 254 nm; Some substances in E. prostrata can be identified at 366 nm; Some substances in A. Rhizoma can be detected in sunlight, with 5% phosphomolybdic acid in ethanol as a chromogenic reagent. The TLC separation was desirable with moderate Rf value and clear spot. The methodology validation for the assay of morroniside, loganin, hyperoside, specnuezhenide, and paeonol presented that they were in good linear correlation in the ranges of 4.432-110.8, 4.192-104.8, 4.040-101.0, 4.132-103.3, and 4.076-101.9 μg/mL, The correlation coefficients of indicator were over 0.999 7. The average recoveries were between 96.57% and 98.67%. The RSD value of intra-day precision was less than 2% and the RSD value of inter-day precision was less than 3%. The method has good stability and reproducibility. Conclusion: The methods of quality control are specific, reproducible, accurate, and suitable, which can be successfully applied to the quality control of ZYG.

HPLC fingerprint and multi-component content determination of Ligustri lucidi Fructus / 中草药

Objective To establish the HPLC fingerpint method for simultaneous determination of four representative components (salidroside, echinacoside, specnuezhenide and oleuropein) of Ligustri Lucidi Fructus, so as to provide a reference for the quality control. Methods The method was performed on an Diamonsil C18 anlytical column (250 mm × 4.6 mm, 5 μm) at the column temperature of 30 ℃. The gradient mobile phase consisted of acetonitrile (A)-0.1% formic acid (B) with a flow rate of 1.0 mL/min. The detection wavelengths were 224 nm. HPLC-Q/TOF-MS were used for identifing the common peaks. Results By studying comparatively the fingerprints of 11 samples, 14 common peaks have been confirmed. There were 12 common peaks were identified by HPLC-Q/TOF-MSE. The similarity of 10 batches are greater than 0.990. Salidroside, echinacoside, specnuezhenide and oleuropein were baseline separated with good linearity relationships (r > 0.999) between concentration and peak areas over the linear ranges. The average recoveries of the investigated compounds were 97.40%, 98.99%, 97.03%, and 100.55%, respectively. Conclusion The method is accurate, reliable and with good reproducibility. It could be used for quality control and evaluation of Ligustri Lucidi Fructus.